Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme.

Glu35 in chicken lysozyme has an abnormally high pKa (6.1) partly due to the hydrophobic environment provided by Trp108. The relationship between protein stability and abnormal pKa was investigated in detail by using mutant lysozymes in which Glu35 was replaced by undissociable residues and an oppositely ionizable residue. It was found that lysozyme was stabilized at alkaline pH range by the replacement of Glu35 with an undissociable residue, Gln (E35Q lysozyme) or Al (E35A lysozyme). On the other hand, when Glu35 was replaced by His (E35H lysozyme), which could have an opposite charge to Glu by ionization, the introduced His35 was found to have an abnormally low pKa (3.6), leading to the destabilization of lysozyme at acidic pH. These observations are completely consistent with the situation that the environment around Glu35 is highly hydrophobic and therefore the placement of either a positive or negative charge in such an environment leads to destabilization of lysozyme. These observations also indicate that the replacement of an acidic residue having abnormally high pKa or a basic residue having abnormally low pKa by an undissociable residue is a very efficient and general method for stabilization of a protein.     

Calorimetric study of the effect of intrachain cross-linking on lysozyme unfolding

Thermodynamics of unfolding of lysozyme cross-linked between Glu 35 and Trp 108 were studied in solutions of various concentrations of 1-propanol (1-PrOH) at pH 3.7 by means of scanning microcalorimetry. The transition temperature for the cross-linked lysozyme increases by 17-19 degrees C due to cross-linking at every concentration of 1-PrOH. This corresponds to the increase in the unfolding Gibbs free energy of about 28 kJ.mol-1, which is independent of the concentration of 1-PrOH. It was found that the unfolding enthalpy of cross-linked lysozyme is only slightly larger than that of intact one, and the unfolding entropy of the cross-linked one is nearly equal to that of the intact one, if both are compared at the same temperature. The stabilization mechanism for the cross-linked lysozyme is discussed on the basis of these calorimetric data.     

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.     

Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes.

The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.     

An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-1 and histidine-15 with bis(bromoacetamide) derivatives by a two-stage reaction procedure and properties of the resulting derivatives

Hen egg white lysozyme was treated at pH 5.5 with four bifunctional reagents, bis(bromoacetamide) derivatives [BrCH2CONH(CH2)nNHCOCH2Br, 1-n, n = 0, 2, 4, and 6], to alkylate His-15 monofunctionally. The excess bifunctional reagent was then removed, and the pH was raised to 9.0 to allow the other end of the reagent molecule to react. The shortest reagent (1-0) gave no intramolecularly cross-linked lysozyme derivative but only histidine-15-modified lysozyme monomer and intermolecularly cross-linked lysozyme dimer. However, the reagents with longer arms (1-2, 1-4, and 1-6) gave lysozyme derivatives cross-linked intramolecularly between the nitrogen at epsilon 2 of His-15 and the epsilon-amino group of Lys-1 without formation of any other intramolecularly cross-linked lysozyme derivative. These results are consistent with our previous proposal that lysozyme has a small hydrophobic pocket that binds small molecules in the direction from His-15 to Lys-1 [Yamada, H., Uozumi, F., Ishikawa, A., & Imoto, T. (1984) J. Biochem. (Tokyo) 95, 503-510]. The thermal stabilities of three cross-linked lysozymes thus obtained were investigated in 0.1 M acetate buffer containing 3 M guanidine hydrochloride at pH 5.5. All derivatives were stabilized but to different degrees. The derivative cross-linked with 1-4 was most stabilized (2.3 kcal/mol), but the derivatives cross-linked with the reagents both shorter (1-2) and longer (1-6) than 1-4 were less stabilized (both 1.6 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of alpha- and beta-N-acetyl-D-glucosamines with hen and turkey lysozymes.

The binding constants of alpha- and beta-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hamaguchi (1975) J. Biochem. 77, 1-16). The pH dependence of the binding of beta-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of beta-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of alpha-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of alpha-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, alpha-GlcNAc binds to hen lysozyme in both alpha- and beta-modes, which correspond to the binding orientation of alpha-GlcNAc and that of beta-GlcNAc, respectively, as determined by X-ray crystallographic studies, but it binds predominantly in the beta-mode below about pH 4, (2) beta-GlcNAc binds to hen and turkey lysozymes predominantly in the beta-mode above about pH 4 and in both alpha- and beta-modes below pH 4, and (3) alpha-GlcNAc binds to turkey lysozyme predominantly in the beta-mode over the whole pH range studied.     

Kinetics of proton transfer reactions of lysozyme with p-nitrophenol near neutral pH—a study of dynamic properties of Glu 35 and His 15

Kinetics of proton transfer between lysozyme and a pH indicator p-nitrophenol (p-Np) were measured by the temperature-jump method in a pH range of 6.0–7.0. Two well-defined relaxation processes were observed. The fast process (τ ? 15 μsec) was also observed for a lysozyme derivative succinylated at the terminal α-amino group of Lys 1. Therefore, the fast process was found to be attributable to the proton transfer reaction of His 15 with p-Np. The slow process (τ ? 50 μsec) was found to be characteristic of the proton transfer reaction of Glu 35, because it disappeared completely in solution containing a lysozyme derivative having an ester crosslink between the carboxyl group of Glu 35 and indol C-2 of Trp 108. The rate constants for proton transfer from Glu 35 and His 15 to p-Np were found to be 9 × 10⁶/sec/M (±65%, 23°C) and 3 × 10⁸/sec/M (±20%, 25°C), respectively. These data indicate that the proton of the carboxyl group of Glu 35 is kinetically stabilized in lysozyme.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.     

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.     

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus

A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (mlDL): Structure-function relationship

Based on the three-dimensional model of the bifunctional enzyme destabilase-lysozyme of the medicinal leech (mlDL) in complex with trimer of N-acetylglucosamine (NAG)₃ by site-directed mutagenesis method, the functional role of the group of amino acids (Glu14, Asp26, Ser29, Ser31, Lys38, His92) in manifestation of lysozyme (glycosidase, muramidase) and isopeptidase activities has been investigated by site-directed mutagenesis. The results obtained go well with hypothesis, that lysozyme active site of mlDL includes catalytic Glu14 and Asp26 residues, and isopeptidase site functions as Ser/Lys catalytic dyad presented by catalytic residues Ser29 and Lys38. Thus, among the invertebrate lysozymes, mlDL presents the first example of a bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

Experimental verification of the crucial roles of Glu73 in the catalytic activity and structural stability of goose type lysozyme

The roles of Glu(73), which has been proposed to be a catalytic residue of goose type (G-type) lysozyme based on X-ray structural studies, were investigated by means of its replacement with Gln, Asp, and Ala using ostrich egg-white lysozyme (OEL) as a model. No remarkable differences in secondary structure or substrate binding ability were observed between the wild type and Glu(73)-mutated proteins, as evaluated by circular dichroism (CD) spectroscopy and chitin-coated celite chromatography. Substitution of Glu(73) with Gln or Ala abolished the enzymatic activity toward both the bacterial cell substrate and N-acetylglucosamine pentamer, (GlcNAc)(5), while substitution with Asp did not abolish but drastically reduced the activity of OEL. These results demonstrate that the carboxyl group of Glu(73) is directly involved in the catalytic action of G-type lysozyme. Furthermore, the stabilities of all three mutants, which were determined from the thermal and guanidine hydrochloride (GdnHCl) unfolding curves, respectively, were significantly decreased relative to those of the wild type. The results obtained clearly indicate the crucially important roles of Glu(73) in the structural stability as well as in the catalytic activity of G-type lysozyme.     

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.     

Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.